Abstract

BACKGROUND AND PURPOSE:

EXPERIMENTAL APPROACH:

The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting.

KEY RESULTS:

NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death.

CONCLUSIONS AND IMPLICATIONS:

Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.

Characterization of NMDA-induced cytotoxicity in SH-SY5Y cultures. (a) Treatment of SH-SY5Y cells with 1.0 mM NMDA for 24 h induces significant cytotoxic effects as assessed by trypan blue staining; no greater cytotoxicity was observed by incubating the cells with a higher concentration (5.0 mM) of the excitotoxin. A lower concentration (0.25 mM) of NMDA did not significantly affect cell viability. Cell death induced by NMDA (1 mM) was prevented by selective, (b) competitive (CGP40116; 100 and 300 μM) and (c) noncompetitive (MK801; 200 nM), antagonists of NMDA subtype of glutamate receptors. (d) 7-Nitroindazole (10 μM), a neuronal NOS preferring inhibitor, prevented death of SH-SY5Y cells induced by NMDA (1.0 mM). Each value in a, b, c and d is the mean±s.e.m. of 3–6 experiments. ***P<0.001 vs control; ###P<0.001 vs NMDA given alone (ANOVA followed by Tukey–Kramer multiple comparisons test). (e) NMDA does not increase caspase-3 activity in SH-SY5Y cell cultures. SH-SY5Y cells were exposed to 1 mM NMDA for the indicated periods of time, and then caspase-3 activity was determined in the supernatants of lysed cell suspensions by measuring cleavage of the fluorogenic substrate Ac-DEVD-AMC (see the Methods section). Results are expressed as relative fluorescence units (RFU) per min per mg of protein. Each value is the mean±s.e.m. of three experiments. *P<0.05 vs control (ANOVA followed by Dunnett multiple comparisons test). (f) Activation of calpain I after exposure of SH-SY5Y cells to NMDA. Representative Western blot showing the increase of the calpain-specific 150/145 kDa SBDP early after NMDA addition (1 mM). Note the lack of accumulation of 120 kDa α-spectrin fragment derived from caspase-mediated proteolysis. Histograms in lower panel show results of densitometric analysis of autoradiographic bands corresponding to 150/145 and 120 kDa SBDP. Data were normalized to the values for α-tubulin. Each value is the mean±s.e.m. of three experiments. *P<0.05 vs control (ANOVA followed by Dunnett multiple comparisons test).

NMDA (1 mM) induced accumulation of intracellular ROS and this was prevented by preincubating SH-SY5Y cells with BEO (0.01%). ROS were measured by fluorescence assay and the data expressed as (Tx−T0/T0) × 100 as described in the Methods section. Each value is the mean±s.e.m. of eight wells per experimental group. Results were confirmed by three independent experiments. *P<0.05 and **P<0.01 vs control, respectively; #P<0.05 and ##P<0.01 vs BEO+NMDA, respectively (ANOVA followed by Tukey–Kramer multiple comparisons test).

Time course of NMDA effects on Akt and GSK-3β phosphorylation in SH-SY5Y cells and protection from NMDA-induced cell death by an inhibitor of GSK-3β kinase. SH-SY5Y cells were exposed to 1 mM NMDA for the indicated periods of time, then cellular proteins were extracted for subsequent analysis by Western blotting of Akt and GSK-3β phosphorylation by using a polyclonal antibody specific for (a) Akt phosphorylated at Ser473 and a polyclonal antibody specific for (c) GSK-3β phosphorylated at Ser9. To establish that changes in phospho-Akt (p-Akt) and phospho-GSK-3β (p-GSK-3β) levels are not due to changes in Akt and GSK-3β protein expression, nitrocellulose membranes were subsequently immunoblotted with an antibody specific for (a) total Akt and (c) total GSK-3β. Equal protein loading in each lane was confirmed by hybridization with an anti-β-actin (a) or anti α-tubulin antibody (c), for Akt anf GSK-3β blots, respectively. Histograms in (b) and (d) show the results of the densitometric analysis of autoradiographic bands; the data are reported as the ratio of (b) p-Akt/total Akt and (d) p-GSK-3β/total GSK-3β, whereas total Akt and total GSK-3β levels were normalized to the values yielded for β-actin and α-tubulin, respectively. Each value is the mean±s.e.m. of 3–6 experiments; *P<0.05 and **P<0.01 vs control, respectively (ANOVA followed by Dunnett multiple comparisons test). (e) MK801 (200 nM; MK), applied to neuroblastoma cultures 10 min before 1 mM NMDA, attenuated reduction of phospho(Ser473)-Akt (p-Akt) levels caused by 5 min exposure to the excitotoxin; a typical example, representative of three independent experiments, is shown. (f) GSK-3 inhibitor IX (0.1 and 1 μM) prevented death of SH-SY5Y cells induced by NMDA. GSK-3 inhibitor IX was added to SH-SY5Y cultures 30 min before NMDA application (1.0 mM) and cell death was evaluated 24 h later by trypan blue staining. No protection was afforded by the enzyme inhibitor applied at a lower concentration (0.001 μM). Each value is the mean±s.e.m. of 3–6 experiments. ***P<0.001 vs control; ###P<0.001 vs NMDA given alone (ANOVA followed by Tukey–Kramer multiple comparisons test).

BEO reduces cell death induced by NMDA in SH-SY5Y cultures. (a) BEO (final concentrations in medium: 0.0005, 0.005 and 0.01%) was added to SH-SY5Y cultures 60 min before application of 1 mM NMDA and cell death was assessed by trypan blue exclusion assay 24 h later; no protection against NMDA-induced cell death was produced by BEO at the lowest concentration (0.0005%). (b) Ethanol (ET, 0.04 and 0.08%), the vehicle used to dissolve BEO, does not affect 1.0 mM NMDA-induced cell death; these concentrations of ethanol correspond to those found in 0.005 and 0.01% BEO. Each value is the mean±s.e.m. of 4–12 experiments. *P<0.05 and ***P<0.001 vs control, respectively; ###P<0.001 vs NMDA given alone (ANOVA followed by Tukey–Kramer multiple comparisons test).

BEO prevented NMDA-induced activation of calpain I. Exposure of SH-SY5Y cells to 1 mM NMDA induced activation of calpain as assessed by Western-blotting analysis of generation of α-spectrin cleavage fragments (150/145 kDa) characteristic of calpain-mediated proteolysis. A significant accumulation of calpain-specific 150/145 kDa SBDP was reached at 5 min after NMDA exposure and this was prevented by a pretreatment (60 min beforehand) with BEO (0.01%). Exposure to NMDA and BEO, given alone or in combination, did not affect generation of 120 kDa α-spectrin fragment derived from caspase-mediated proteolysis. Histograms in lower panel show results of densitometric analysis of autoradiographic bands corresponding to 150/145 and 120 kDa SBDP. Data were normalized to the values yielded for α-tubulin. Each value is the mean±s.e.m. of three experiments. *P<0.05 vs control and #P<0.05 vs NMDA given alone (ANOVA followed by Tukey–Kramer multiple comparisons test).

BEO reduced NMDA-induced decrease of phospho-Akt and phospho-GSK-3β levels in SH-SY5Y cells. (a). Exposure of SH-SY5Y cells to 1 mM NMDA for 2 and 5 min induced deactivation of Akt kinase as determined by Western-blot analysis of phospho(Ser473)-Akt (p-Akt) levels and this was attenuated by BEO (0.01%) applied to neuroblastoma cultures 60 min before NMDA exposure. (b) Densitometric analysis of immunoreactive bands shows that the significant changes in phospho(Ser473)-Akt levels induced by BEO in NMDA-treated cells were not associated with changes in total Akt immunoreactivity. (c) Exposure of SH-SY5Y cells to 1 mM NMDA for 5 but not for 2 min induced activation of GSK-3β kinase as determined by Western-blot analysis of phospho(Ser9)-GSK-3β (p-GSK-3β) levels and this is attenuated by BEO (0.01%) applied to neuroblastoma cultures 60 min before NMDA addition. As confirmed by densitometric analysis of immunoreactive bands (d), BEO did not affect the expression of total GSK-3β in NMDA-treated cells. Each value in b and d is the mean±s.e.m. of 3–6 experiments; *P<0.05 and **P<0.01 vs control, respectively (ANOVA followed by Tukey–Kramer multiple comparisons test).

BEO restored Akt and GSK-3β phosphorylation, reduced by serum withdrawal, via a PI3K-dependent mechanism. SH-SY5Y cells were maintained in serum-containing medium (+serum) or cultured in serum-free medium for 1 h (−serum); serum-starved cells were exposed to 0.01% BEO for 5, 10, 20, 60 and 120 min (min), then cellular proteins were extracted for subsequent analysis by Western blotting of Akt and GSK-3β phosphorylation by using polyclonal antibodies specific for Akt phosphorylated at Ser473 or for GSK-3β phosphorylated at Ser9. Serum deprivation caused a dramatic decline in phospho-Akt (p-Akt) and phospho-GSK-3β (p-GSK-3β) immunoreactivity. Application of BEO for 5–20 min enhanced phosphorylation of Akt and GSK-3β reduced by serum deprivation; this effect peaked at 10 min, then it progressively declined. A preincubation for 30 min with the specific PI3K inhibitor, LY294002 (LY; 20 μM), before exposure to BEO for 10 min, prevented the effects of the essential oil on Akt and GSK-3β phosphorylation. Representative immunoblots from three independent experiments are shown.

Monoterpene hydrocarbons could be involved in the neuroprotection afforded by BEO against NMDA-induced cell death. SH-SY5Y cultures were incubated with a bergapten-free extract of the essence (BEO-BF (a)) or with a fraction of the oil deprived of bergapten and of monoterpene hydrocarbons (BEO-BF/HF (b)) for 60 min before application of 1 mM NMDA and cell death was assessed by trypan blue exclusion assay 24 h later. (a) BEO-BF, but not (b) BEO-BF/HF, concentration dependently reduced NMDA-induced cell death. Each value is the mean±s.e.m. of 4–8 experiments. ***P<0.001 vs control; ###P<0.001 vs NMDA given alone (ANOVA followed by Tukey–Kramer multiple comparisons test).